DNA methods.

Isolation of plasmid DNA and genomic DNA, restriction endonuclease digestions, agarose gel electrophoresis, and DNA ligations were performed using standard methods (52). Restriction enzymes, T4 DNA ligase, Taq polymerase, and high-fidelity Phusion polymerase were purchased from Promega Corp. (Madison, WI), New England Biolabs (Beverly, MA), or MJ Research (Waltham, MA) and were used according to the manufacturers' instructions.

Construction of bacterial strains.

To create an in-frame (nonpolar) deletion in the B. bronchiseptica fhaS gene, an allelic exchange system employing the sacB gene was used as previously described (2, 39). Initially, an allelic exchange plasmid, pEG7SΔfhaS, was constructed as follows. Primers EBPSFHA3x (5′-AATCTAGAACCCGCAACCAGTCCGAGAAGTACG-3′) and EBPSFHA4x (5′-AAGGTACCCGCGTTCATGTTGTCTCCTGATGTCG-3′) were used to amplify a 672-bp fragment corresponding to the 5′ region and containing the first three codons of the fhaS coding sequence. Primers were designed such that XbaI and KpnI sites are introduced at the 5′ and 3′ ends of the PCR product, respectively. Primers EBPSFHA1x (5′-AAGGTACCCTGCATCGCTAGCGCGAGG-3′) and EBPSFHA2 (5′-AAGCATGCCGCAGGACATCGACATAGGG-3′) were used to amplify a 679-bp fragment including the last three codons of the fhaS coding sequence and downstream sequences. Primers were designed such that KpnI and SphI sites were introduced at the 5′ and 3′ ends of the PCR product, respectively. These fragments were purified, ligated into TA cloning vectors (Invitrogen), digested with the appropriate restriction enzymes, and cloned into BamHI-XbaI-digested pEG7S, a derivative of pEG7 (2), resulting in an in-frame deletion of all but the first three and last three codons of the fhaS gene. Sequence analysis of the cloned DNA (Laragen, Los Angeles, CA) was performed to confirm the fidelity of B. bronchiseptica sequences. The resulting plasmid (pEG7SΔfhaS) was transformed into E. coli SM10 λpir and then mobilized into the RB50 chromosome by conjugation. Cointegrants were selected on medium containing streptomycin (B. bronchiseptica strains are naturally streptomycin resistant) and gentamicin. Recombinants that had excised the plasmid from the chromosome were selected on media containing 10% sucrose (the sacB gene encodes for levansucrase, which is toxic to cells grown in the presence of high concentrations of sucrose) (19), and PCR was used to screen for mutants that had undergone a recombination event resulting in a chromosomal deletion of the fhaS gene. A ΔfhaB ΔfhaS double mutant was constructed by the same method by mobilizing pEG7SΔfhaS into RBX9.

To construct strains that harbor chromosomal lacZ reporter plasmids or fhaC null mutations, pEG110, pEG111, or pSJ22 was transformed into SM10 λpir and then conjugated into the appropriate B. bronchiseptica strain. Cointegrants were selected on media containing streptomycin and gentamicin. PCR was used to verify integration of the plasmid at the proper chromosomal locus. For strains containing pEG111, PCR was used to distinguish between integration events at either the fhaB or fhaS locus, since the 1.7-kb fhaB fragment in pEG111 is nearly identical to sequences present in the fhaS gene.

β-Galactosidase assays.

Stationary phase cultures from strains harboring lacZ transcriptional fusions were used to measure β-galactosidase activity. Bacteria were grown overnight in either LB broth or SS media and permeabilized by the addition of sodium dodecyl sulfate (SDS) and CHCl₃, and β-galactosidase activity was determined essentially as described previously (39), except that measurements were taken using a Victor³ 1420 microplate reader (PerkinElmer Life Sciences, Boston, MA).

Bacterial cell fractionation and immunoblot analysis.

Stationary-phase cultures were used for all bacterial cell preparations. Whole-cell lysates were prepared as described previously (39). Outer membrane preparations were isolated from bacterial cell sonicates as the Triton X-100 insoluble fraction. Briefly, clarified cell sonicates were spun at 16,000 × g, the pellet was resuspended in 2% Triton X-100 and spun again, and the pellet was resuspended in an appropriate volume of 10 mM HEPES (pH 7.5). Secreted proteins were concentrated by addition of 10% TCA to the culture supernatant and then recovered by centrifugation. Proteins were resuspended in an equal mixture of 1× SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer (52) and saturated Tris solution (to neutralize excess acid).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli (38) using denaturing 3 to 8% linear gradient gels (acrylamide-bisacrylamide, 29:1). Proteins from whole cells (derived from approximately 8.0 × 10⁸ CFU), outer membranes (derived from 5.0 × 10⁹ CFU), or supernatant fractions (derived from 1.5 × 10¹⁰ CFU) were run on SDS-PAGE, transferred to nitrocellulose membranes (Schleicher and Schuell BioScience, Dassel, Germany), and incubated with a 1:10,000 dilution of chicken polyclonal FHA antibody (raised against amino acids [aa] 1048 to 1385 of B. bronchiseptica FHA, which are identical [except for one amino acid difference] with the corresponding sequence in FhaS). Antigen-antibody complexes were detected by incubating with a 1:20,000 dilution of goat anti-chicken IRdye 800-conjugated secondary antibody (Rockland, Gilbertsville, PA) and visualized using the Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE).

In vitro adherence assay.

Adherence to rat lung epithelial (L2) cells was performed as described previously (12), with the exception that LB broth was used as the bacterial growth medium as well as the diluent for addition of bacteria to cell monolayers.

In vitro competition experiment.

Wild-type (RB50G) and ΔfhaS strains were grown overnight in either SS or LB and then diluted by coinoculating equivalent numbers of each (based on measurements of optical density at 600 nm) into fresh media to an optical density at 600 nm of approximately 0.1. Cultures were then incubated on a roller at 37°C for 8 h (approximately five cell divisions). The relative proportion of wild-type to ΔfhaS bacteria was determined by plating aliquots onto media with and without gentamicin.

In vivo competition experiment.

Three- to 4-week-old female Wistar rats (Charles River Laboratories, Wilmington, MA) were coinoculated intranasally with approximately 250 CFU of the wild-type and 250 CFU of the ΔfhaS strain (from cultures grown overnight in SS media) essentially as previously described (41). Animals were sacrificed 7, 14, and 28 days postinoculation, and colonization levels in the nasal cavity and trachea were determined as described previously (41).

Phylogenetic analysis of fhaS alleles.

Total genomic DNA was prepared from various B. bronchiseptica strains, B. pertussis strain Tohama I, and B. parapertussis_hu strain 12822 and used as templates for PCR. Primers PfhaSOUT1 (5′-TGGCGGCCCGCTATATCGA-3′) and CD64 (5′-GGCTGAGCGGGCCGCGCTGG-3′) were used to amplify B. bronchiseptica RB50-specific fhaS sequences, and primers PfhaSOUT1 and fhaSBpBpp1086.rev (5′-GCCGGCATGCAGCGACAAGGCGTC-3′) were used to amplify fhaS sequences specific to B. pertussis and B. parapertussis_hu. Cycling conditions were as follows: 94°C for 2 min followed by 30 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 2 min, with a final extension step of 72°C for 10 min.

Expression of fhaS.

Expression of all Bordetella virulence genes characterized so far is positively regulated by the BvgAS phosphorelay and is maximal in cells grown at 37°C in SS broth (Bvg⁺ phase conditions) and minimal in cells grown at room temperature in SS broth or at 37°C in SS broth supplemented with ≥20 mM MgSO₄ or nicotinic acid (Bvg⁻ phase or “modulating” conditions). Expression of the Bvgⁱ phase gene bipA is both positively and negatively regulated by BvgAS; it is maximal in cells grown at intermediate temperatures in SS broth or at 37°C in SS broth containing “semimodulating” concentrations of MgSO₄ or nicotinic acid (Bvgⁱ phase conditions) (71). To determine the expression pattern of fhaS in B. bronchiseptica, we constructed a strain containing a chromosomal fhaS-lacZ fusion and measured β-galactosidase activity in cultures grown at 37°C ± 50 mM MgSO₄. fhaS-lacZ expression was approximately 2.5-fold greater in cells grown in SS broth than in cells grown in SS broth + MgSO₄. This expression pattern was similar in profile, but not magnitude, to Bvg-activated genes such as fhaB and cyaA (Fig. ​(Fig.2).2). To determine whether fhaS expression is controlled by BvgAS, we introduced the fhaS-lacZ fusion into Bvg⁺ and Bvg^- phase-locked derivatives of RB50. Expression of fhaS-lacZ in the Bvg⁺ phase-locked strain grown in SS broth with or without added MgSO₄ was similar to fhaS-lacZ expression in the wild-type strain grown in SS broth. fhaS-lacZ expression was the same in the Bvg^- phase-locked strain grown in SS ± MgSO₄ as in the wild-type strain grown in SS + MgSO₄ (Fig. ​(Fig.2).2). These results demonstrate that fhaS is positively regulated by BvgAS.

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FIG. 2.

Comparison of fhaS expression patterns with those of known Bvg-regulated genes. An fhaS::lacZ, fhaB::lacZ, bipA::lacZ, or cyaA::lacZ transcriptional fusion was introduced into the chromosome of wild type (WT), Bvg⁺ phase locked (Bvg^c), Bvgⁱ phase locked (Bvgⁱ) or Bvg⁻ phase locked (Bvg⁻) B. bronchiseptica strains as indicated. Bacteria were grown overnight in Stainer-Scholte (SS) or L broth (LB) in the presence (+) or absence (-) of 50 mM MgSO₄. β-galactosidase activity is reported in Miller units, and data represent the average of three independent experiments done in duplicate. Error bars indicate one standard deviation from the mean.

We discovered serendipitously that fhaS expression is approximately fivefold greater in cells grown in LB broth than in cells grown in SS broth (Fig. ​(Fig.2).2). Since we had previously observed that wild-type B. bronchiseptica showed differences in colony morphology and motility when grown on LB agar, consistent with modulation to the Bvgⁱ or Bvg^- phase (not shown), we hypothesized that LB might be semimodulating. Consistent with this hypothesis, expression of the gene encoding adenylate cyclase, cyaA, (which is minimal under Bvgⁱ phase conditions) (11), was decreased in LB-grown cultures compared with SS-grown cultures, and expression of bipA (which is maximal under Bvgⁱ phase conditions) (18) was slightly greater in LB-grown cultures than in SS-grown cultures (Fig. ​(Fig.2).2). These results suggested that fhaS, like bipA, might be expressed maximally under Bvgⁱ phase conditions. However, fhaS-lacZ expression was greater in the Bvg⁺ phase-locked strain than in the Bvgⁱ phase-locked strain, and expression in both of these strains was greater when cells were grown in LB than in SS (Fig. ​(Fig.2).2). fhaS therefore does not appear to be a Bvgⁱ phase gene like bipA (compare fhaS-lacZ expression with bipA-lacZ expression in the various strain backgrounds and media in Fig. ​Fig.2).2). Instead, fhaS appears to be expressed differently than all previously characterized Bvg-regulated genes; it is expressed maximally only under a subset of Bvg-activating conditions. Inspection of the 246-nucleotide sequence between the end of the gene upstream of fhaS (purL) and the fhaS initiation codon revealed no discernible BvgA binding sites, suggesting Bvg-mediated control of fhaS expression is indirect.

Cellular localization of FhaS.

In Bordetella strains grown in vitro, mature FHA can be detected in concentrated culture supernatants, outer membrane fractions, and whole-cell lysates, while preprocessed FhaB can be detected only in whole-cell lysates. Because of the extensive similarity between FhaS and FhaB, we hypothesized that FhaS is processed and localized in a manner similar to FhaB. To test this hypothesis, we performed Western blot analysis on proteins derived from whole-cell, outer membrane, and supernatant fractions from wild-type, ΔfhaB, ΔfhaS, and ΔfhaB ΔfhaS B. bronchiseptica strains grown overnight in either LB or SS. Membranes were probed with an anti-FHA antibody generated against amino acids 1048 to 1385 of B. bronchiseptica FHA, a region surrounding the RGD motif in which FhaS and FHA are predicted to differ by only one amino acid. In whole-cell lysates, this antibody detected many polypeptides, with one of ~350 kDa and one of ~260 kDa being most prominent in the wild-type and ΔfhaS strains grown in either LB or SS (Fig. ​(Fig.3).3). Because only the highest-molecular-weight polypeptides were detected when an antibody against the C terminus of FhaB was used (data not shown), we can conclude that the ~350-kDa and ~250-kDa polypeptides correspond to unprocessed FhaB and mature FHA, respectively. In whole-cell lysates of the wild-type and ΔfhaB strains, prominent polypeptides of approximately 230 to 240 kDa were detected when the cells were grown in LB. Similarly sized polypeptides were only minimally detectable when these strains were grown in SS (Fig. ​(Fig.3).3). The absence of these polypeptides in lysates of the ΔfhaB ΔfhaS double mutant strongly suggests that they are FhaS. Cleavage within the amino acids that are identical to those in FhaB that are predicted to represent the C-terminal maturation site would result in maturation of the 328-kDa full-length FhaS protein into a mature protein of ~233 kDa. It therefore appears that the 230-to-240-kDa polypeptides present in lysates of the wild-type and ΔfhaB strains, but absent in the ΔfhaS and ΔfhaS ΔfhaB strains, represent a processed, mature form of FhaS.

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FIG. 3.

FhaS localizes to the outer membrane and is released into the culture supernatant. Proteins from whole cell (A), outer membrane (B), and supernatant (C) fractions were prepared from the wild-type (WT) and the indicated mutant strains grown in either LB or Stainer-Scholte (SS) broth and subjected to immunoblot analysis using a polyclonal antibody to B. bronchiseptica FHA, which also recognizes FhaS. The positions of molecular weight markers (in kDa) are shown.

In outer membrane preparations, the anti-FHA antibody detected distinct polypeptides from 230 to 260 kDa in the wild-type and ΔfhaS strains grown in SS, corresponding to FHA. Polypeptides with the same mobility were present in these strains grown in LB; however, several additional prominent polypeptides ranging in size from ~180 to 240 kDa were detected in the wild-type and ΔfhaB strains grown in this medium (but not in SS). Because these polypeptides were absent in the ΔfhaB ΔfhaS double mutant, they most likely correspond to FhaS. Moreover, these data suggest that, like FHA, several distinct forms of FhaS are localized to the outer membrane, or alternatively, there is significant proteolysis of FHA and FhaS in outer membrane preparations.

The anti-FHA antibody also detected several polypeptides in concentrated culture supernatants. At least five polypeptides ranging from ~150 to 260 kDa were detected in supernatants prepared from the wild-type and ΔfhaS strains when the cells were grown in SS (Fig. ​(Fig.3).3). The top three bands were also present in these strains when grown in LB, indicating that FHA is efficiently secreted from the cell under both growth conditions. In addition, polypeptides of ~230 to 240 kDa were detected in the wild-type and ΔfhaB strains when the cells were grown in LB (and were only minimally detected from the ΔfhaB strain when the cells grown in SS), demonstrating that FhaS is also secreted from the cell, and in amounts similar to FHA when the cells are grown in LB.

Export of FhaS is dependent on FhaC.

FhaC is an outer membrane channel-forming protein required for the export of FhaB to the cell surface (22, 31, 33, 51). Sequences within FhaB required for FhaC-dependent export are located within the N-terminal 300 aa (the so-called “secretion domain”) (7, 36). Although the first ~150 aa of FhaS and FhaB are not identical, they are moderately similar and two amino acid motifs (NPGL and NPNG) that have been shown to be critical for export of FhaB (32) are present in the same relative location in FhaS. Moreover, a search of the B. bronchiseptica genome revealed no other open reading frames with similarity to FhaC. We therefore hypothesized that FhaS is exported via FhaC. Previous experiments have shown that FhaB/FHA is undetectable in strains containing loss-of-function mutations in fhaC, presumably because it is degraded in the periplasm (32, 70). Using Western blot analysis, we found that polypeptides corresponding to FHA and/or FhaS were detectable in whole-cell lysates of the wild-type, ΔfhaB, and ΔfhaS strains grown in LB but were undetectable in lysates of strains containing disruption mutations in fhaC (Fig. ​(Fig.4).4). These results demonstrate that like FHA, FhaS is dependent on FhaC, and strongly suggest that FhaS is exported across the outer membrane by FhaC. B. bronchiseptica, therefore, appears to use the same protein for the export of both FHA and FhaS.

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FIG. 4.

Export of FhaS is dependent on FhaC. Whole cell lysates were prepared from LB-grown wild-type (WT) and ΔfhaS, ΔfhaB, and ΔfhaB ΔfhaS mutant strains in isogenic fhaC⁺ (+) and fhaC⁻ (−) backgrounds. Proteins were subjected to immunoblot analysis and probed with anti-FHA antibody, which also recognizes FhaS. The positions of molecular mass markers (in kDa) are shown.

Evaluation of FhaS-mediated adherence of B. bronchiseptica to epithelial cells.

The role of B. pertussis FHA as an adhesin is well documented (4, 25, 28, 43, 44, 49, 50, 54, 59-61). Additionally, it has been demonstrated that FHA is both necessary and sufficient to mediate B. bronchiseptica adherence to a rat lung epithelial (L2) and other cell lines in vitro (12, 41). FhaS shares a high degree of similarity with FHA in regions that have been implicated in mediating adherence to eukaryotic cells or cell surface components, including the CRD (>99% identical), which directs binding to glycolipids and ciliated cells (49), and the HBD (99% identical), which promotes binding to highly sulfated polysaccharides, structures commonly found on eukaryotic cell surfaces (23). The integrin-binding RGD motif is also conserved between FhaS and FHA. To test the hypothesis that FhaS can mediate binding of B. bronchiseptica to eukaryotic cells, we utilized L2 cells in a standard in vitro adherence assay with a ΔfhaB strain grown overnight in LB in order to induce maximal expression of FhaS in the absence of FHA. Under these growth conditions, FhaS is produced and localized to the cell surface in amounts similar to FHA (Fig. ​(Fig.3).3). Although wild-type RB50 grown in LB adhered efficiently to the L2 cells (Fig. ​(Fig.5A),5A), the ΔfhaB strain was severely defective in adherence (Fig. ​(Fig.5B),5B), indicating that, at least under these experimental conditions, FhaS was not able to mediate binding of B. bronchiseptica to L2 cells. To determine whether FhaS contributes to adherence in the presence of FHA (i.e., that both FHA and FhaS are required for adherence of LB-grown bacteria to epithelial cells), we compared the binding ability of the ΔfhaS strain with that of wild-type B. bronchiseptica. The ΔfhaS strain adhered to L2 cells as efficiently as RB50, suggesting that binding of the wild-type strain to L2 cells, even when grown in LB, is mediated primarily by FHA (Fig. ​(Fig.5C).5C). We obtained similar results using a human bronchial epithelial (BEAS-2B) cell line (data not shown). FhaS, therefore, does not appear to be important for mediating adherence to epithelial cells—at least not in a manner that we can detect with these cultured cell assays.

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FIG. 5.

FhaS does not mediate adherence of B. bronchiseptica to rat lung epithelial (L2) cells. Bacteria were grown overnight in LB and added to a monolayer of L2 cells in a standard adherence assay. L2 cells were incubated with wild-type RB50 (A); ΔfhaB (B); ΔfhaS (C); or L broth alone (D). Data are representative of similar results obtained from three independent experiments. Magnification is ×1,000.

Role of FhaS in respiratory tract infection.

To evaluate the contribution of fhaS to respiratory tract infection, we performed both single-infection and coinfection experiments. When inoculated into separate animals, the ΔfhaS strain did not differ from RB50 in its ability to colonize the nasal cavities and tracheas of Wistar rats at days 14 and 28 post infection (data not shown). However, when coinoculated into the same animal with RB50G, a derivative of our wild-type strain expressing a gentamicin resistance gene (6), a defect for the ΔfhaS strain was revealed. In these experiments, groups of rats were inoculated intranasally with 5 μl of phosphate-buffered saline containing approximately 250 CFU each of wild-type and mutant bacteria, and the number of wild-type and mutant bacteria recovered from the nasal cavities and tracheas of infected animals was determined 7, 14, and 28 days postinoculation. The competitive index (CI) was calculated as the ratio of mutant to wild-type bacteria recovered divided by the ratio of mutant to wild-type bacteria administered; thus, a CI of less than 1 indicates a virulence defect for the mutant strain. On days 7 and 14 postinoculation, although the total CFU recovered (shown at the top of each panel in Fig. ​Fig.6)6) was comparable to that from animals infected with wild-type bacteria alone, the ΔfhaS strain was significantly outcompeted by the wild-type strain in both the nasal cavity and trachea (Fig. ​(Fig.6).6). By day 28, however, although in the majority of animals the number of CFU of the ΔfhaS mutant recovered was far lower than that seen with the wild-type strain, in a few cases the number of ΔfhaS CFU isolated was close to or higher than the number of wild-type CFU isolated. The ΔfhaS strain was not outcompeted by RB50G when coinoculated into SS or LB broth and grown for several generations in vitro (see Materials and Methods). Together, these data indicate that fhaS contributes to bacterial fitness in vivo in a manner revealed when the ΔfhaS strain must compete with wild-type bacteria.

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FIG. 6.

ΔfhaS strains are out-competed by wild-type B. bronchiseptcia in vivo. Wistar rats were inoculated intranasally with approximately 250 CFU of both the wild-type (RB50G) and ΔfhaS strains. Animals were sacrificed on the indicated day postinoculation, and the number of wild-type and ΔfhaS bacteria present in the nasal cavity and trachea was determined. Each symbol represents the competitive index (CI) from the indicated tissue of a single animal, which was calculated as the ratio of mutant to wild-type bacteria recovered divided by the ratio of mutant to wild-type bacteria inoculated. A CI value of 1 (thin dashed line) indicates equal numbers of wild-type and ΔfhaS bacteria were recovered. Symbols below the thick dashed line represent cases where no ΔfhaS bacteria were present in at least 500 total colonies recovered. The average number of CFU recovered from each tissue ± 1 standard error of the mean (log₁₀) is shown at the top of each panel. *, P < 0.05.

We thank Carol Inatsuka for helpful discussions and technical assistance, Robin Hulbert for critical review of the manuscript, and Joseph Mazar for technical assistance.

This research was supported by grants from the National Institutes of Health (AI43896), U.S. Department of Agriculture (National Research Initiative of the USDA Cooperative State Research, Education and Extension Service grant 2003-35204-13555), and the University of California Superfund Basic Research and Education Program to P.A.C.